Tumors are heterogeneous tissues that contain a small population of stem-like cells that self-renew, differentiate into various progeny types, and survive hostile microenvironments to form tumors. Such tumor- initiating cells (TICs) have been identified in cell lines and patient samples using surface markers and their ability to generate tumor spheres and xenograft tumors. However, TIC sub-populations from various sources differ greatly in their surface marker expression profile, and, to date, there is no universal marker profile to identify TICs. This inability to effectively isolate TIC sub-populations with high purity/yield is a profound impediment to characterizing the biology of these cells as well as analyzing patient biopsies for effective diagnosis or prognosis. We recently established a platform technology, micro Stem cell High-Efficiency Adhesion-based Recovery (SHEAR), to isolate human pluripotent stem cells and differentiated progeny based on differences in adhesive forces in a label-free, rapid (<10 min), and efficient (>95percent purity, >95percent yield) manner using microfluidics. This technology was developed to purify pluripotent stem cells and progeny, but our preliminary data support its capacity to enrich TICs. The objective of this Innovative Technologies for Cancer-Relevant Biospecimen Science R21 project is to establish an integrated SHEAR-cell profiling microfluidic platform to purify TIC sub- populations from heterogeneous cell lines and tumors. Our central hypothesis is that sub-populations of TICs exhibit distinct `adhesive force signatures' that can be exploited to selectively purify them with high efficiency using SHEAR. Aim 1: Establish the ability of the integrated microfluidics platform to purify TIC sub- populations from normal and cancer mammary cell lines based on adhesive force signatures. Aim 2: Examine the ability of the integrated microfluidics platform to purify TIC sub-populations from xenograft tumors. The proposed research is highly innovative because it integrates unique and state-of-the-art bioengineering technologies to purify TIC sub-populations from heterogeneous cell lines and xenograft tumors. This project is expected to yield several outcomes. First, we will establish whether TIC sub-populations can be purified by differences in adhesive force signature and how the adhesive force signature correlates to surface marker profile and tumorigenicity. Second, we will establish the ability of the integrated microfluidics platform to purify and identify TIC sub-populations from xenograft tumors. This innovative research will establish a broadly applicable, easily implemented, and robust technology to purify and characterize TIC sub-populations for basic studies of cancer heterogeneity and to maximize the quality and utility of samples derived from biospecimens for downstream diagnostic and prognostic analyses.